The last decade has seen an increase in the understanding of the critical role nitric oxide as a blood vessel dilator contributing to the regulation of blood flow and cardiovascular homeostasis. Nitric oxide may be oxidized in blood to nitrite (NO2—), an anion considered to be an inert metabolic end product of such nitric oxide oxidation. In vivo plasma levels of nitrite have been reported to range from 150 to 1000 nM, and the nitrite concentration in aortic ring tissue has been reported to be in excess of 10,000 nM (Rodriguez et al., Proc Natl Acad Sci USA, 100, 336-41, 2003; Gladwin et al., Proc Natl Acad Sci USA, 97, 9943-8, 2000; and Rassaf et al., Nat Med, 9, 481-3, 2003). This potential storage pool for NO is in excess of plasma S-nitrosothiols, which have been reported to be less than 10 nM in human plasma (Rassaf et al., Nat Med, 9, 481-3, 2003; Rassaf et al., Free Radic Biol Med, 33, 1590-6, 2002; Rassaf et al., J Clin Invest, 109, 1241-8, 2002; and Schechter et al., J Clin Invest, 109, 1149-51, 2002). Mechanisms have been proposed for the in vivo conversion of nitrite to NO, for example, by enzymatic reduction by xanthine oxidoreductase or by non-enzymatic disproportionation/acidic reduction (Millar et al., Biochem Soc Trans, 25, 528S, 1997; Millar et al., FEBS Lett, 427, 225-8, 1998; Godber et al., J Biol Client, 275, 7757-63, 2000; Zhang et al., Biochem Biophys Res Commun, 249, 767-72, 1998 [published erratum appears in Biochem Biophys Res Commun 251, 667, 1998]; Li et al., J Biol Chem, 276, 24482-9, 2001; Li et al., Biochemistry, 42, 1150-9, 2003; Zweier et al., Nat Med, 1, 804-9, 1995; Zweier et al., Biochim Biophys Acta, 1411, 250-62, 1999; and Samouilov et al., Arch Biochem Biophys, 357:1-7, 1998).
Arterial-to-venous gradients of nitrite across the human forearm at rest and during regional NO synthase inhibition have been observed, with increased consumption of nitrite occurring with exercise (Gladwin et al., Proc Natl Acad Sci USA, 97, 9943-8, 2000; Gladwin et al, Proc Natl Acad Sci USA, 97, 11482-11487, 2000; and Cicinelli et al., Clin Physiol, 19:440-2, 1999). Kelm and colleagues have reported that large artery-to-vein gradients of nitrite form across the human forearm during NO synthase inhibition (Lauer et al., Proc Natl Acad Sci USA, 98, 12814-9, 2001). Unlike the more simple case of oxygen extraction across a vascular bed, nitrite may be both consumed, as evidenced by artery-to-vein gradients during NO synthase inhibition and exercise, and produced in the vascular bed by endothelial nitric oxide synthase-derived NO reactions with oxygen.
At high concentrations, nitrite has been reported to be a vasodilator in vitro (Ignarro et al., Biochim Biophys Acta, 631, 221-31, 1980; Ignarro et al., J Pharmacol Exp Ther, 218, 739-49, 1981; Moulds et al., Br J Clin Pharmacol, 11, 57-61, 1981; Gruetter et al., J Pharmacol Exp Ther, 219, 181-6, 1981; Matsunaga et al., J Pharmacol Exp Ther, 248, 687-95, 1989; and Laustiola et al., Pharmacol Toxicol, 68, 60-3, 1991). The levels of nitrite shown to vasodilate in vitro have always been in excess of 100,000 nM (100 μM) and usually at millimolar concentrations.
Consistent with the high concentrations of nitrite required to vasodilate in vitro, when Lauer and colleagues infused nitrite into the forearm circulation of human subjects, they reported no vasodilatory effects, even with concentrations of 200 μM in the forearm (Lauer et al., Proc Natl Acad Sci USA, 98, 12814-9, 2001). Lauer et al. reported that a “complete lack of vasodilator activity of intraarterial infusions of nitrite clearly rules out any role for this metabolite in NO delivery” and concluded that “physiological levels of nitrite are vasodilator-inactive.” Furthermore, Rassaf and colleagues also failed to find a vasodilatory effect in humans following infusion of nitrite (Rassaf et al., J Clin Invest, 109, 1241-8, 2002). Thus, in vivo studies have concluded that physiological levels of nitrites do not serve as a source for NO, and that physiological levels of nitrites do not have a role in regulating blood pressure.
Historically, nitrite has been used as a treatment for cyanide poisoning. High concentrations are infused into a subject suffering cyanide poisoning in order to oxidize hemoglobin to methemoglobin, which will bind cyanide. These high concentrations of nitrite produce clinically significant methemoglobinemia, potentially decreasing oxygen delivery. While these high concentrations of nitrite have been shown to decrease blood pressure in humans, the amount of methemoglobin formed precluded a use for nitrite in the treatment of other medical conditions.
Therefore, the state of the art was that nitrite was not a significant vasodilator at concentrations below 100 μM in vitro, and even when infused into humans at concentrations of 200 μM in the forearm. It was also the state of the art that nitrite was not converted to nitric oxide in the human blood stream.